In prokaryotes, DNA replication proceeds bidirectionally from a single specific initiation site. In eukaryotes DNA replication is bidirectional from multiple initiation sites. We intend to determine whether eukryotic initiation sites are specific and whether their temporal sequence of activation is constant by preparing a replication map of a yeast chromosomal DNA. This will be done by electron microscopic observation of a circular derivative of yeast chromosome III. A yeast cell cycle mutant, cdc 2, fails to replicate approximately 20% of its genome at the nonpermissive temperature. We will characterize the fraction of the genome that fails to replicate to determine whether cdc 2 is defective in replicating specific DNA sequences. We will isolate mutants of yeast defective in metochondrial DNA replication.